TY - JOUR
T1 - Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
AU - Si, Tong
AU - Li, Bin
AU - Comi, Troy J.
AU - Wu, Yuwei
AU - Hu, Pingfan
AU - Wu, Yuying
AU - Min, Yuhao
AU - Mitchell, Douglas A.
AU - Zhao, Huimin
AU - Sweedler, Jonathan V.
N1 - Funding Information:
We gratefully acknowledge financial support from the National Institutes of Health (GM077596 to H.Z., AI113219 to J.V.S., and GM097142 to D.A.M.). J.V.S. also acknowledges NSF CHE 16-067915. T.S. acknowledges postdoctoral fellowship support from the Carl R. Woese Institute for Genomic Biology (UIUC). T.J.C. acknowledges support from an NSF Graduate Research Fellowship Program, the Springborn Fellowship, and the Training Program at the Chemistry-Biology Interface (T32 GM070421). We thank Prof. Joshua D. Shrout at the University of Notre Dame for kindly providing P. aeruginosa genomic DNA. We thank Elizabeth Neumann for the help with microscopy and FT-ICR analyses, Sage J.B. Dunham for initiating the rhamnolipid engineering project, and Adam J. DiCaprio with the construction of PZN analogue libraries. We also thank Dr. Benjamin Bowen at the Lawrence Berkeley National Lab for the help with mass spectrum deposition at OpenMSI.
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/9/13
Y1 - 2017/9/13
N2 - Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) imaging has been used for rapid phenotyping of enzymatic activities, but is mainly limited to single-step conversions. Herein we report a label-free method for high-throughput engineering of multistep biochemical reactions based on optically guided MALDI-ToF MS analysis of bacterial colonies. The bacterial cells provide containment of multiple enzymes and access to substrates and cofactors via metabolism. Automated MALDI-ToF MS acquisition from randomly distributed colonies simplifies procedures to prepare strain libraries without liquid handling. MALDI-ToF MS profiling was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. For analogues of the peptidic antibiotic plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding were used to group similar spectra for rapid identification of nonisobaric variants. After MALDI-ToF MS screening, follow-up analyses using high-resolution MS and tandem MS were readily performed on the same sample target. Separately, relative ion intensities of rhamnolipid congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate the recovery of desirable mutants. For both the antibiotic and rhamnolipid cases, large populations of colonies were rapidly surveyed at the molecular level, providing information-rich insights not easily obtained with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-ToF MS instruments, this simple yet effective workflow is applicable for a wide range of screening campaigns targeting multistep enzymatic reactions.
AB - Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) imaging has been used for rapid phenotyping of enzymatic activities, but is mainly limited to single-step conversions. Herein we report a label-free method for high-throughput engineering of multistep biochemical reactions based on optically guided MALDI-ToF MS analysis of bacterial colonies. The bacterial cells provide containment of multiple enzymes and access to substrates and cofactors via metabolism. Automated MALDI-ToF MS acquisition from randomly distributed colonies simplifies procedures to prepare strain libraries without liquid handling. MALDI-ToF MS profiling was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. For analogues of the peptidic antibiotic plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding were used to group similar spectra for rapid identification of nonisobaric variants. After MALDI-ToF MS screening, follow-up analyses using high-resolution MS and tandem MS were readily performed on the same sample target. Separately, relative ion intensities of rhamnolipid congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate the recovery of desirable mutants. For both the antibiotic and rhamnolipid cases, large populations of colonies were rapidly surveyed at the molecular level, providing information-rich insights not easily obtained with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-ToF MS instruments, this simple yet effective workflow is applicable for a wide range of screening campaigns targeting multistep enzymatic reactions.
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U2 - 10.1021/jacs.7b04641
DO - 10.1021/jacs.7b04641
M3 - Article
C2 - 28792758
AN - SCOPUS:85029485794
VL - 139
SP - 12466
EP - 12473
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 36
ER -